U.S. patent application number 10/482791 was filed with the patent office on 2004-12-09 for process for producing nicotinamide adenine dinucleotide phosphate(nadp).
Invention is credited to Ando, Yoshio, Kawai, Shigeyuki, Matsuo, Yuhsi, Murata, Kousaku, Tomisako, Shoichi.
Application Number | 20040248263 10/482791 |
Document ID | / |
Family ID | 19038369 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248263 |
Kind Code |
A1 |
Kawai, Shigeyuki ; et
al. |
December 9, 2004 |
Process for producing nicotinamide adenine dinucleotide
phosphate(nadp)
Abstract
The present invention provides a novel process for preparing
nicotinamide adenine dinucleotide phosphate (NADP). The process of
the present invention comprises performing phosphorylation using a
polyphosphoric acid or a salt thereof and nicotinamide adenine
dinucleotide (NAD.sup.+) as substrates in the presence of a
polyphosphate-dependent NAD.sup.+ kinase from a Mycobacterium,
wherein the reaction solution contains 0.1-15% by weight of the
polyphosphoric acid or a salt thereof, and 5-150 mM of a divalent
metal ion.
Inventors: |
Kawai, Shigeyuki; (Kyoto,
JP) ; Murata, Kousaku; (Kyoto, JP) ; Tomisako,
Shoichi; (Tokyo, JP) ; Ando, Yoshio; (Tokyo,
JP) ; Matsuo, Yuhsi; (Tokyo, JP) |
Correspondence
Address: |
Nixon & Vanderhye
8th Floor
1100 North Glebe Road
Arlington
VA
22201-4714
US
|
Family ID: |
19038369 |
Appl. No.: |
10/482791 |
Filed: |
July 20, 2004 |
PCT Filed: |
July 2, 2002 |
PCT NO: |
PCT/JP02/06692 |
Current U.S.
Class: |
435/89 |
Current CPC
Class: |
C12P 19/36 20130101 |
Class at
Publication: |
435/089 |
International
Class: |
C12P 019/30 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 2, 2001 |
JP |
2001201400 |
Claims
1. A process for preparing nicotinamide adenine dinucleotide
phosphate (NADP) comprising performing phosphorylation using a
polyphosphoric acid or a salt thereof and nicotinamide adenine
dinucleotide (NAD+) as substrates in the presence of a
polyphosphate-dependent NAD+ kinase from a Mycobacterium, wherein
the reaction solution contains 0.1-15% by weight of the
polyphosphoric acid or a salt thereof, and 5-150 mM of a divalent
metal ion.
2. The process of claim 1 wherein the NAD+ kinase from a
Mycobacterium is NAD+ kinase from M. tuberculosis (Mycobacterium
tuberculosis).
3. The process of claim 1 wherein the NAD+ kinase from a
Mycobacterium is selected from: 1) a polypeptide having the amino
acid sequence of SEQ ID NO: 1, or 2) a polypeptide having an amino
acid sequence having deletion, addition or substitution of one or
more amino acid residues in the amino acid sequence of SEQ ID NO: 1
while having polyphosphate-dependent NAD+ kinase activity.
4. The process of claim 3 wherein the NAD+ kinase from a
Mycobacterium is a polypeptide having the amino acid sequence of
SEQ ID NO: 1.
5. The process of claim 1 wherein the NAD+ kinase from a
Mycobacterium is used as a solubilized or immobilized natural or
recombinant protein or as immobilized cells.
6. The process of claim 1 wherein the reaction solution contains
2-10% by weight of a polyphosphoric acid or a salt thereof.
7. The process of claim 1 wherein the polyphosphoric acid or a salt
thereof is selected from the group consisting of metaphosphoric
acid, hexametaphosphoric acid and salts thereof and mixtures
thereof.
8. The process of claim 1 wherein the reaction solution contains
50-100 mM of a divalent metal ion.
9. The process of claim 1 wherein the divalent metal ion is
selected from magnesium ion or manganese ion.
10. The process of claim 1 wherein the divalent metal ion is
contained as a metal salt selected from a chloride, sulfate or
nitrate in the reaction solution.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a process for preparing
nicotinamide adenine dinucleotide phosphate (NADP).
BACKGROUND ART
[0002] NADP (nicotinamide adenine dinucleotide phosphate) has been
used as a diagnostic reagent for enzymatically analyzing blood and
urine [1]. Reactions for transferring phosphates to NAD.sup.+
(nicotinamide adenine dinucleotide) have been previously performed
by enzymatic synthesis reactions using NAD.sup.+ kinases, which are
often derived from microorganisms. For example, NAD.sup.+ kinases
from microorganisms, such as Brevibacteria and Corynebacteria as
well as from yeasts and animals and plants are described in "Enzyme
Handbook" 1983, Asakura Publishing, p.339 [20]; and Matsushita H et
al.1986, Can. J. Microbiol. 32:585-590 [2].
[0003] NAD.sup.+ kinases are classified into the following three
main categories according to the type of phosphate donor to
NAD.sup.+. The relation with industrial utility is summarized in
the table below.
1TABLE 1 Substrate specificity Utility for NAD.sup.+ Type of the
phosphate donor synthesis 1 ATP x (ATP is very expensive) 2 Both
polyphosphate and AT 3 Polyphosphate
[0004] At present, industrial production of NADP relies on
enzymatic processes including ATP-dependent NAD kinases
(EC2.7.1.23) catalyzing phosphorylation of NAD in the presence of
ATP [2]. This is partially because most of NAD.sup.+ kinases are
ATP-dependent NAD.sup.+ kinases (type 1 in the table above) which
are widely present in microorganisms, yeasts, animals and plants so
that they are readily available for industrial applications.
NADP.sup.+ synthesis using ATP-dependent NAD.sup.+ kinases must be
coupled to ATP regeneration reaction because industrially expensive
ATP is used. The balance between ATP regeneration reaction and
NADP.sup.+ synthesis reaction is summarized by the formulae below.
1 NAD + + ATP NADP + + ADP ADP + X - P ATP + X = NAD + + X - P NADP
+ + X
[0005] X-P: a high energy phosphate compound in living bodies, e.g.
acetyl phosphate, carbamyl phosphate, phosphoenol pyruvate, ADP,
etc.
[0006] X: acetic acid, carbamic acid, pyruvic acid, AMP, etc.
[0007] Enzymes used for ATP regeneration reaction: acetate kinase,
carbamate kinase, pyruvate kinase, adenylate kinase, etc.
[0008] However, the above processes have such disadvantage as
expensive ATP and low stability and cellular contents of the
enzymes. Thus, NAD.sup.+ kinases capable of utilizing inexpensively
available polyphosphates as phosphate donors are desirable for
industrially producing NADP.sup.+ from NAD.sup.+. Polyphosphates
are polymers of inorganic orthophosphate residues linked via
inorganic phosphate bonds energetically equivalent to the phosphate
bonds of ATP (FIG. 1) [6]. Polyphosphates are commercially
available in larger amounts at very lower cost as compared with
ATP.
[0009] Polyphosphate-dependent NAD kinases (types 2 and 3 in Table
1) have already been reported by Murata K. et al. (Biotechnol.
Bioeng., 1979 21:887-895; and Agric. Biol. Chem., 1980 44:61-68)
[4], but they have not been industrially applied because of the low
cellular contents thereof in the reported Brevibacterium. This may
be attributed to the low activity of polyphosphate NAD kinases in
cells of B. ammoniagenes.
[0010] On the other hand, Kawai et al. (Biochem, Biophys. Res.
Commun., 276, pp. 57-63 (2000)) [7] describes that an open reading
frame of unknown function Rv1695 from M. tuberculosis
(Mycobacterium tuberculosis) of the genus Mycobacterium, H37Rv
encodes a polyphosphate-dependent NAD.sup.+ kinase. However,
optimal reaction conditions for preparing NADP have not been
examined well, and development of processes for more efficiently
and inexpensively preparing NADP have been demanded.
DISCLOSURE OF THE INVENTION
[0011] An object of the present invention is to provide a novel
process for preparing nicotinamide adenine dinucleotide phosphate
(NADP). The process of the present invention comprises performing
phosphorylation using a polyphosphoric acid or a salt thereof and
nicotinamide adenine dinucleotide (NAD.sup.+) as substrates in the
presence of a polyphosphate-dependent NAD.sup.+ kinase from a
Mycobacterium, characterized in that the reaction solution contains
0.1-15% by weight of the polyphosphoric acid or a salt thereof, and
5-150 mM of a divalent metal ion.
[0012] In an embodiment of the process of the present invention, an
NAD.sup.+ kinase from M. tuberculosis (Mycobacterium tuberculosis)
is preferably used.
[0013] In an embodiment of the process of the present invention,
the reaction solution contains 2-10% by weight of a polyphosphoric
acid or a salt thereof. Preferably, the reaction solution contains
50-100 mM of a divalent metal ion.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the structure of inorganic polyphosphate
[poly(P)] having a chain length of n+2.
[0015] FIG. 2 shows the NADP-producing activity of the purified
Ppnk protein. The method described in (2) Assay for NADP-producing
activity in "Materials and Methods" below was applied unless
otherwise indicated.
[0016] (A) shows the effect of the concentration of metaphosphoric
acid on the NADP-producing activity of purified Ppnk. The
NADP-producing activity was assayed in the presence of 50 (solid
squares), 100 (solid circles) or 150 (solid triangles) mg/ml of
metaphosphoric acid.
[0017] (B) shows the effect of the concentrations of NADP and ADP
on the NADP-producing activity of purified Ppnk. The NADP-producing
activity was assayed in the presence of various levels of NADP
(solid circles) or ADP (open circles).
[0018] FIG. 3 shows functions of Ppnk in acetone-treated
immobilized cells. The functions of Ppnk in the acetone-treated
immobilized cells were evaluated by measuring the NADP-producing
activity as compared with the activity in the non-immobilized
acetone-treated cells. The method described in (2) Assay for
NADP-producing activity in "Materials and Methods" was applied
unless otherwise indicated. The maximum NADP-producing activity of
Ppnk in each cell preparation was assumed to be 100%.
[0019] (A) Thermostability
[0020] The acetone-treated immobilized cells (solid circles) or the
acetone-treated cells (open circles) were incubated in 0.5 ml of
5.0 mM Tris-HCl (pH 7.0) for 10 minutes at various temperatures.
Persistence of the NADP-producing activity was determined.
[0021] (B) Optimal Temperature
[0022] The acetone-treated immobilized cells (solid circles) or the
acetone-treated cells (open circles) were incubated at various
temperatures to determine persistence of the NADP-producing
activity.
[0023] (C) Optimal pH
[0024] The acetone-treated immobilized cells (solid triangles and
solid circles) or the acetone-treated cells (open triangles and
open circles) were incubated in 100 mM sodium acetate (solid
triangles and open triangles) or Tris-HCl (solid circles and open
circles) to determine persistence of the NADP-producing
activity.
[0025] (D) Stability After use
[0026] The acetone-treated immobilized cells (solid circles) or the
acetone-treated cells (open circles) were repeatedly used for
assaying NADP-producing activity. After each run of reaction for 1
hour, cells were washed in 5.0 mM Tris-HCl (pH 7.0) and reused for
further reaction in a freshly prepared reaction mixture.
[0027] FIG. 4 shows functions of Ppnk in the acetone-treated
immobilized cells. The procedure for acetone treatment and the
assay for the NADP-producing activity of Ppnk in the cells were as
described in 2) Assay for NADP-producing activity in "Materials and
Methods" unless otherwise indicated. However, the amounts of (A)
metaphosphoric acid, (B) ATP, (C) NAD and (D) Mg.sup.2+ were
changed. In (A), precipitates were formed at concentrations lower
than 40 mg/ml metaphosphoric acid. In (B), ATP was used in place of
polyphosphate. The maximum NADP-producing activity of Ppnk was
assumed to be 100%.
[0028] FIG. 5 shows NADP production by the acetone-treated
immobilized SK27 cells (solid line) and SK45 cells (dotted line).
The method described in (2) Assay for NADP-producing activity in
"Materials and Methods" was applied unless otherwise indicated. A
reaction for producing NADP was performed in an optimal reaction
mixture (4.0 ml ) consisting of 50 mM NAD, 100 mM MgCl.sub.2, 100
mM Tris-HCl (pH 7.0), immobilized cells (0.10 g), and 100 mg/ml
metaphosphoric acid (A) or 150 mM ATP (B). At each time indicated,
10 .mu.l of the reaction mixture was recovered and assayed for NADP
level in the mixture.
[0029] FIG. 6 shows analytic results of the reaction products.
Purified Ppnk (B and E) and the immobilized cells (C and F) were
used for a reaction for producing NADP in the presence of ATP (left
column) or metaphosphoric acid (right column). Changes of
components were tested before (A and D) and after (B, C, E, and F)
the reaction.
[0030] FIG. 7 shows elution patterns of phosphate donors in
metaphosphoric acid on a Dowex 1.times.2 column. The dialysate
(outer solution containing phosphate polymers) was loaded onto a
Dowex 1.times.2 column and adsorbed phosphate polymers were eluted
with a linear gradient of 0-1.0 M (pH 2.0) LiCl. Solid circles
represent phosphate-donating activity, open circles represent
acid-labile phosphate, and the dotted line represents the
concentration of LiCl.
DETAILED DESCRIPTION OF THE INVENTION
[0031] As a result of intensive studies to solve the above
problems, we found a process for efficiently and inexpensively
preparing NADP to accomplish the present invention.
[0032] Therefore, the present invention provides a novel process
for preparing nicotinamide adenine dinucleotide phosphate (NADP).
The process of the present invention comprises performing
phosphorylation using a polyphosphoric acid or a salt thereof and
nicotinamide adenine dinucleotide (NAD.sup.+) as substrates in the
presence of a polyphosphate-dependent NAD.sup.+ kinase from a
Mycobacterium, characterized in that the reaction solution contains
0.1-15% by weight of the polyphosphoric acid or a salt thereof, and
5-150 mM of a divalent metal ion.
[0033] NAD is a coenzyme involved in oxidation-reduction (redox)
reactions and its reduced form is NADH. It is also called
diphosphopyridine nucleotide (DPN, DPNH), coferment, coenzyme I
(CoI), etc. and it is the most abundant coenzyme found in living
bodies. NAD has a structure consisting of nicotinamide
mononucleotide (NMN) and adenylic acid linked via a phosphodiester
bond. The oxidized form of NAD is called NAD.sup.+ because the
nitrogen atom in the pyridine ring exists as a pyridinium ion.
NAD.sup.+ is biosynthetically produced from a precursor such as
tryptophan mainly in the liver in animals or glycerol and aspartate
in plants through quinolinic acid. It is also synthesized from
vitamins, i.e. nicotinic acid and nicotinamide. Important functions
of NAD.sup.+ result from the NAD.sup.+ reduction coupled to the
biological energy producing mechanism (oxidation reaction).
[0034] NADP was discovered as a coenzyme acting on
glucose-6-phosphate dehydrogenase during studies of
glucose-6-phosphate metabolism in erythrocytes. NADP basically
shares the structure of NAD and contains an additional phosphate
attached to the 2'-position of the ribose of adenylic acid in NAD
via an ester bond. NADP abundantly occurs in the liver in living
bodies but at about a half of the level of NAD. The conversion
manner between the oxidized and reduced forms of NADP is similar to
that of NAD, and oxidized NADP is positively charged by a
pyridinium ion (NADP.sup.+). NADP and NAD resemble each other in
structure and reaction manner, but they are strictly distinguished
by enzymes. NADP is involved in reactions catalyzed by
glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase,
L-glutamate dehydrogenase, etc. These enzymatic reactions are
widely used as indicator reactions of various coupled enzyme
reactions as well as for spectroscopic quantitative assays of NADP.
The assays are typically based on the difference in the absorbance
A.sub.340 of reduced NADP (NADPH). More sensitive assays can be
achieved by treating NADP.sup.+ with an alkali to convert into a
fluorescent derivative.
[0035] Reactions for synthesizing biological components such as
fatty acids and steroids involve several steps of reduction using
NADPH as a major hydrogen donor. In contrast, not NADP.sup.+ but
NAD.sup.+ and flavin protein (FP) are involved in oxidation
processes as found in decomposition systems-energy production
systems (glycolysis, citrate pathway, .beta.-oxidation of fatty
acids). NADP and NAD clearly exist in reciprocal redox states and
have distinct functions in cells.
NAD.sup.+ kinases
[0036] NAD.sup.+ kinases are enzymes transferring phosphate groups
of substrates to the 2'-position of the ribose of adenylic acid of
NAD.sup.+ to produce NADP.sup.30 . Conventionally, ATP has been
normally used as a phosphate-donating substrate. NAD.sup.30 kinases
used in the processes of the present invention are characterized as
polyphosphate-dependent NAD.sup.+ kinases utilizing polyphosphate
or both polyphosphate and ATP as phosphate-donating substrate. In
other words, the process of the present invention does not use ATP
which was conventionally used as a phosphate donor. Thus, the
reaction mixture solution is free from ADP or AMP, byproducts due
to the conventional use of ATP during phosphate transfer reactions
catalyzed by NAD.sup.+ kinases. Thus, high-purity NADP.sup.+ can be
obtained in an NAD.sup.+ kinase-mediated reaction mixture
solution.
[0037] Polyphosphates are polymers of inorganic orthophosphate
residues linked via inorganic phosphate bonds energetically
equivalent to the phosphate bonds of ATP as shown in FIG. 1 (FIG.
1) [6]. In FIG. 1, n represents the degree of condensation.
Preferably, n is, but not limited to, 3 to 32. Relative
reactivities of NAD.sup.+ kinases from M. tuberculosis
(Mycobacterium tuberculosis) of the present invention and that from
Micrococcus flavus as a control in relation to the degree of
condensation of polyphosphate are described in detail by Kawai et
al. (Biochem. Biophys. Res. Commun., 276, pp.57-63 (2000)) [7].
Polyphosphates are commercially available in larger amounts at very
lower cost as compared with ATP. Preferred polyphosphates used in
the present invention include, but not limited to, metaphosphoric
acid, hexametaphosphoric acid and salts thereof. Metaphosphoric
acid and salts thereof are preferred. Polyphosphates are available
from for example, Wako Pure Chemical Industries, Sigma-Aldrich,
Merck, etc.
[0038] Metaphosphoric acid is also called glacial phosphoric acid
and represented by general formula (HPO.sub.3).sub.n. Normally, it
exists as a polymer such as trimer or tetramer of cyclic or linear
polymetaphosphoric acids. As used herein, "metaphosphoric acid"
also includes such trimer or tetramer cyclic or linear
polymetaphosphoric acids. Polymetaphosphoric acid is a cyclic
compound formed of phosphate groups linked by anhydride bonds and
represented by general formula H.sub.nP.sub.nO.sub.3n. It is a
viscous liquid at room temperature, but becomes a glass-like solid
solution upon cooling. Aqueous solutions are acidic and readily
decomposed into orthophosphoric acid upon heating. It is hydrolyzed
by metaphosphatase to open the ring into polyphosphoric acid. In
the living world, high molecular polymers are found in bacteria,
fungi and algae or the like, while metaphosphoric acids having low
degrees of condensation such as trimetaphosphoric acid are found in
yeasts and some bacteria. The phosphate anhydride linkage is the
so-called high energy phosphate linkage. Reactions catalyzed by
polyphosphate kinases are reversible and seem to be used for high
energy phosphate linkage and phosphate storage.
[0039] Polyphosphate-Dependent NAD.sup.+ Kinases from a
Mycobacterium
[0040] The NAD.sup.+ kinases of the present invention are derived
from a Mycdbacterium such as M. tuberculosis (Mycobacterium
tuberculosis), M. leprae, M. bovis, M. avium, M. paratuberculosis,
M. smegmatis, M. chlorophenolicum, M. diernhoferi, M. forluitum, M.
phlei and M. vaccae. Detailed descriptions of strains belonging to
the genus Mycobacterium can be found in e.g. Institute for
Fermentation (http://www.ifo.or.jp) or American Type Culture
Collection (http://www.atcc.org).
[0041] The NAD.sup.+ kinases of the present invention utilize
polyphosphate or both polyphosphate and ATP as phosphate-donating
substrate. They preferably have a reactivity to polyphosphate of
60% or more, more preferably 80% or more, still more preferably
100% or more, most preferably 120% or more as compared with the
reactivity to ATP.
[0042] The NAD.sup.+ kinase proteins of the present invention are
not limited to any sources or preparation processes so far as they
have characteristics described herein. That is, the NAD.sup.+
kinase proteins of the present invention may be any of those
natural proteins, or expressed from recombinant DNAs by genetic
engineering techniques, or chemically synthesized. Alternatively,
the purified proteins may be used in an immobilized state or cells
expressing said proteins may be used in an immobilized state in the
present invention, as described below.
[0043] The NAD.sup.+ kinases from the genus Mycobacterium of the
present invention are preferably derived from M. tuberculosis
(Mycobacterium tuberculosis). A typical NAD.sup.+ kinase herein is
a protein having the amino acid sequence of SEQ ID NO:1 consisting
of amino acid residues No.1 -No.307. SEQ ID NO:1 is an amino acid
sequence deduced from the nucleotide sequence of an open reading
frame of unknown function Rv1695 from M. tuberculosis
(Mycobacterium tuberculosis) of the genus Mycobacterium, H37Rv
described by Kawai et al. (Biochem. Biophys. Res. Commun., 276, pp.
57-63 (2000)) [7]. The genomic fragment Rv1695 has been deposited
with The Sanger Center (see http://www.sanger.ac.uk/Projects/M
tuberculosis/cosmids.stml). The NAD.sup.+ kinase from Mycobacterium
or the NAD.sup.+ kinase from M. tuberculosis is hereinafter
sometimes referred to as "Ppnk (Polyphosphate-dependent NAD
kinase)".
[0044] Another currently known NAD.sup.+ kinase from Mycobacteria
is a sequence from M. leprae. Specifically, Blast searches based on
the primary amino acid sequence of Ppnk protein from M.
tuberculosis (Rv1695: Mycobacterium tuberculosis H37Rv) revealed
homology to the known gene sequences. The results are shown
below.
Homology to the Primary Amino Acid Sequence of Ppnk
[0045]
2 Microorganisms Homology Mycobacterium tuberculosis 100%
Mycobacterium leprae 93% Streptomyces coelicolor 70%
[0046] It is well known that naturally occurring proteins include
variant proteins having one or more amino acid changes resulting
from e.g. the presence of a genetic variations caused by different
species or ecotypes producing them. As used herein, the term "amino
acid change" means to include substitutions, deletions, insertions
and/or additions of one or more amino acids. The protein of the
present invention typically has the amino acid sequence of SEQ ID
NO:1 based on the presumption from the nucleotide sequence of the
gene. However, it is not limited to only the protein having this
sequence, but intended to encompass any homologous proteins having
characteristics defined herein. For example, proteins lacking a
part of the amino acid sequence of SEQ ID NO:1 can be used for the
preparation process of the present invention so far as they have
the property of utilizing polyphosphate or both polyphosphate and
ATP as phosphate-donating substrates. The "amino acid change"
involves one or more amino acids, preferably 1-20, more preferably
1-10, most preferably 1-5 amino acids.
[0047] Thus, the NAD.sup.+ kinases from the genus Mycobacterium of
the present invention include polypeptides having an identity of at
least 70% or more to SEQ ID NO:1 and polyphosphate-dependent
NAD.sup.+ kinase activity. The identity is at least 70% or more,
preferably 75% or more, more preferably 80% or more, still more
preferably 90% or more, most preferably 95% or more.
[0048] The percent identity may be determined by visual inspection
and mathematical calculation. Alternatively, the percent identity
of two protein sequences can be determined by comparing sequence
information using the GAP computer program, based on the algorithm
of Needleman and Wunsch (J. Mol. Bio., 48:443 (1970)) and available
from the University of Wisconsin Genetics Computer Group (UWGCG).
The preferred default parameters for the GAP program include: (1) a
scoring matrix, blosum62, as described by Henikoff et al. (Proc.
Natl. Acad. Sci. USA, 89:10915 (1992)); (2) a gap weight of 12; (3)
a gap length weight of 4; and (4) no penalty for end gaps.
[0049] Other programs used by those skilled in the art of sequence
comparison may also be used. The percent identity can be determined
by comparing sequence information using the BLAST program described
by Altschul et al. (Nucl. Acids. Res. 25, pp. 3389-3402, 1997), for
example. This program is available at the website of National
Center for Biotechnology Information (NCBI) or DNA Data Bank of
Japan (DDBJ) on the Internet. Various conditions (parameters) for
homology searches with the BLAST program are described in detail on
the site, and searches are normally performed with default values
though some settings may be appropriately changed.
[0050] Generally, modified proteins containing a change from one to
another amino acid having similar properties (such as a change from
a hydrophobic amino acid to another hydrophobic amino acid, a
change from a hydrophilic amino acid to another hydrophilic amino
acid, a change from an acidic amino acid to another acidic amino
acid or a change from a basic amino acid to another basic amino
acid) often have similar properties to those of the original
protein. Methods for preparing such recombinant proteins having a
desired variation using genetic engineering techniques are well
known to those skilled in the art and such variant proteins are
also included in the scope of the present invention.
[0051] The present invention further includes polypeptides with or
without associated native-pattern glycosylation. Polypeptides
expressed in yeast or mammalian expression systems (e.g., COS-1 or
COS-7 cells) may be similar to or significantly different from a
native polypeptide in molecular weight and glycosylation pattern,
depending upon the choice of an expression system. Expression of
polypeptides of the invention in bacterial expression systems, such
as E. coli, provides non-glycosylated molecules. Further, a given
preparation may include multiple differentially glycosylated
species of the protein. Glycosyl groups can be removed through
conventional methods, in particular those utilizing glycopeptidase.
In general, glycosylated polypeptides of the invention can be
incubated with a molar excess of glycopeptidase (Boehringer
Mannheim).
Processes for Preparing NAD.sup.+ Kinase Proteins
[0052] The Ppnk protein of the present invention may be purified
from M. tuberculosis strain H37Rv, for example, according to known
procedures. Cells of M. tuberculosis H37Rv can be dissolved in an
appropriate buffer (that can be selected from phosphate buffers
having buffer capacity in a pH range of 6-8 such as Tris-HCl buffer
and various Good's buffers) and then successively fractionated by
molecular sieve (gel filtration) chromatography, Blue affinity
chromatography, anion exchange chromatography and hydrophobic
chromatography to give a pure sample. During purification steps,
the NAD.sup.+ kinase activity determined by a known method can be
used as an indicator.
[0053] Alternatively, said protein can be obtained in mass by
genetic engineering techniques by transducing a DNA sequence
containing nucleic acid residues No. 1-No. 921 of SEQ ID NO:2
encoding Ppnk of SEQ ID NO:1 or a part thereof into E. coli, yeasts
or cells of insect or certain animal cells using an expression
vector capable of being amplified in each host and expressing the
DNA sequence.
[0054] The amino acid sequence of the human NAD.sup.+ kinase
protein and the DNA sequence encoding it are disclosed herein as
SEQ ID NOs:1 and 2. They can be wholly or partially used to readily
isolate a gene encoding a protein having a similar physiological
activity from other species using genetic engineering-techniques
including hybridization and nucleic acid amplification reactions
such as PCR. In such cases, the proteins encoded by such genes can
also be used in the present invention.
[0055] Hybridization conditions used for screening homologous genes
are not specifically limited, but stringent conditions are
generally preferred, such as 6.times.SSC, 5.times.Denhardt's
solution, 0.1% SDS at 25-68.degree. C. The hybridization
temperature here is more preferably 45-68.degree. C. (without
formamide) or 25-50.degree. C. (50% formamide). It is well known to
those skilled in the art that DNAs containing a nucleotide sequence
having a homology equal to or higher than a certain level can be
cloned by appropriately selecting hybridization conditions such as
formamide level, salt level and temperature, and all of thus cloned
homologous genes are included in the scope of the present
invention.
[0056] Nucleic acid amplification reactions here include reactions
involving temperature cycles such as polymerase chain reaction
(PCR) (Saiki et al., 1985, Science, 230, pp. 1350-1354), ligase
chain reaction (LCR) (Wu et al., 1989, Genomics, 4, pp. 560-569;
Barringer et al., 1990, Gene, 89, pp. 117-122; Barany et al., 1991,
Proc. Natl. Acad. Sci. USA, 88, 189-193) and transcription-based
amplification (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA, 86,
pp. 1173-1177) as well as isothermal reactions such as strand
displacement amplification (SDA) (Walker et al., 1992, Proc. Natl.
Acad. Sci. USA, 89, pp. 392-96; Walker et al., 1992, Nuc. Acids
Res., 20, pp. 1691-1696), self-sustained sequence replication (3SR)
(Guatelli et. al., 1990, Proc. Natl. Acad. Sci. USA, 87, pp.
1874-1878), and Q.beta. replicase system (Lizardi et al., 1988,
BioTechnology, 6, pp. 1197-1202). Other reactions such as nucleic
acid sequence-based amplification (NASBA) using competitive
amplification of a target nucleic acid and a variant sequence
disclosed in European Patent No. 0525882 can also be used. PCR is
preferred.
[0057] Homologous genes cloned by hybridization or nucleic acid
amplification reactions as above have an identity of at least 70%
or more, preferably 80% or more, more preferably 90% or more, most
preferably 95% or more to the nucleotide sequence shown as SEQ ID
NO:2 in the Sequence Listing.
[0058] The percent identity may be determined by visual inspection
and mathematical calculation. Alternatively, the percent identity
of two nucleic acid sequences can be determined by comparing
sequence information using the GAP computer program, version 6.0
described by Devereux et al. (Nucl. Acids Res., 12:387 (1984)) and
available from the University of Wisconsin Genetics Computer Group
(UWGCG). The preferred default parameters for the GAP program
include: (1) a unitary comparison matrix (containing a value of 1
for identities and 0 for non-identities) for nucleotides, and the
weighted comparison matrix of Gribskov and Burgess, Nucl. Acids
Res., 14:6745 (1986), as described by Schwartz and Dayhoff, eds.,
Atlas of Protein Sequence and Structure, National Biomedical
Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps. Other programs used by one
skilled in the art of sequence comparison may also be used.
[0059] Recombinant vectors for integrating a gene to express a
protein herein can be prepared by known methods. Methods for
integrating a DNA fragment of the gene of the present invention
into a vector such as a plasmid are described in e.g. Sambrook, J.
et al, Molecular Cloning, A Laboratory Manual (2nd edition), Cold
Spring Harbor Laboratory, 1.53 (1989). Commercially available
ligation kits (e.g. available from Takara Bio Inc.) can be
conveniently used. Thus obtained recombinant vectors (e.g.
recombinant plasmids) are transferred into host cells (e.g. E. coli
JM109, BL21(DE3)pLysS, TB1, LE392 or XL-1Blue), preferably JM109
(e.g. available from Takara Bio Inc.), BL21(DE3)pLysS (Novagen,
Darmstadt, Germany).
[0060] Suitable methods for introducing a plasmid into a host cell
include the Hanahan method (Hanahan, D.,J. Mol. Biol., 166: pp.
557-580 (1983)) or the use of calcium phosphate or calcium
chloride/rubidium chloride, electroporation, electroinjection,
chemical treatment with PEG or the like, the use of a gene gun
described in Sambrook, J. et al., Molecular Cloning, A Laboratory
Manual (2nd edition), Cold Spring Harbor Laboratory, 1.74
(1989).
[0061] Vectors can be conveniently prepared by linking a desired
gene by a standard method to a recombination vector available in
the art (e.g. plasmid DNA). Specific examples of suitable vectors
include, but are not limited to, E. coli-derived plasmids such as
pET3a (Novagen), pTRP (Japanese Patent Public Disclosure
No.103278/96), pBluescript, pUC18, pUC19, pBR322, preferably pET3a
or pTRP. In constructing expression vectors, dicistronic systems
may be used for rapid transcription to mRNAs. Detailed descriptions
of dicistronic systems are found in e.g. Brigitte E. et al., Method
in Enzymology 185: pp. 94-103, 1990 [21]. A combinations of the
vector pTRP with a dicistronic system, i.e. the vector pTRP-2cis
can also be used.
[0062] As a preferred embodiment of the present invention, a
transformant pET3a-NADK/BL21(DE3)pLysS obtained by transducing an
expression vector pET3a-NADK containing the ppnk gene into a host
cell BL21(DE3)pLysS was deposited on Jun. 20, 2001 with the
International Patent Organism Depositary (IPOD) of the National
Institute of Advanced Industrial Science and Technology (residing
at Tsukuba Central 6, 1-1-1 Higashi, Tsukuba-city,
Ibaraki-prefecture, 305-8566, Japan) under FERM P-18383. Similarly,
a transformant pTRP-2cis-NADK/JM109 obtained by transducing an
expression vector pTRP-2cis-NADK containing the ppnk gene into a
host cell JM109 was deposited on Jun. 20, 2001 with the
International Patent Organism Depositary of the National Institute
of Advanced Industrial Science and Technology (residing at Tsukuba
Central 6, 1-1-1 Higashi, Tsukuba-city, Ibaraki-prefecture,
305-8566, Japan) under FERM P-18384.
[0063] Expression vectors are especially useful for the purpose of
producing a desired protein. The types of expression vectors are
not specifically limited so far as they can express a desired gene
in various prokaryotic and/or eukaryotic host cells to produce a
desired protein. Preferred known vectors include expression vectors
for E. coli such as pQE-30, pQE-60, pMAL-C2, pMAL-p2, pSE420;
expression vectors for yeasts such as pYES2 (genus Saccharomyces),
pPIC3.5K, pPIC9K, pAO815 (all genus Pichia); and expression vectors
for insects such as pBacPAK8/9, pBK283, pVL1392, pBlueBac4.5.
[0064] An example of an expression vector for use in mammalian host
cells is a vector constructed as disclosed by Okayama and Berg
(Mol. Cell. Biol. 3:280 (1983)). A useful system for stable high
level expression of mammalian cDNAs in C127 murine mammary
epithelial cells can be constructed substantially as described by
Cosman et al. (Mol. Immunol. 23:935 (1986)). Alternatively,
suitable vectors for in vivo or in vitro expression in nerve cells
include adenovirus vectors or a modified vector (pEF-CITE-neo,
Miyata, S et al., Clin. Exp. Metastasis, 16: pp. 613-622, 1998) of
pEF-BOS vector (Mizushima, S. et al., Nucl. Acid Res. 18: p. 5322,
1990).
[0065] Transformants can be prepared by introducing a desired
expression vector into a host cell. Suitable host cells are not
specifically limited so far as they are compatible with the
expression vector and can be transformed therewith, and include
various cells such as naturally occurring cells or artificially
established recombinant cells commonly used in the technical field
of the present invention. Examples are bacteria (Escherichia,
Bacillus), yeasts (Saccharomyces, Pichia), animal cells, insect
cells, plant cells, etc.
[0066] Host cells are preferably E. coli, yeasts or insect cells,
specifically E. coli such as M15, JM109, BL21; yeasts such as
INVSc1 (the genus Saccharomyces), GS115, KM71 (all the genus
Pichia); insect cells such as BmN4, silkworm larva. Examples of
animal cells are those derived from mouse, Xenopus, rat, hamster,
simian or human or culture cell lines established from these cells.
Plant cells include those derived from tobacco, Arabidopsis, rice,
maize, wheat, etc., but are not specifically limited so far as they
can be cell-cultured.
[0067] When a bacterium, especially E. coli is used as a host cell,
the expression vector generally consists of at least a
promoter/operator region, a start codon, a gene encoding a desired
Ppnk protein, a stop codon, a terminator and a replicable unit.
[0068] When a yeast, plant cell, animal cell or insect cell is used
as a host cell, the expression vector generally preferably contains
at least a promoter, a start codon, a gene encoding a desired Ppnk
protein, a stop codon and a terminator. It may also contain a DNA
encoding a signal peptide, an enhancer sequence, non-translated 5'
and 3' regions of the desired gene, a selectable marker or a
replicable unit, etc., if desired.
[0069] A preferred start codon in vectors of the present invention
is a methionine codon (ATG). A stop codon may be conventional stop
codons (e.g. TAG, TGA, TAA).
[0070] The replicable unit refers to a DNA capable of replicating
the whole DNA sequence in a host cell, and means to include natural
plasmids, artificially modified plasmids (plasmids prepared from
natural plasmids) and synthetic plasmids, etc. Preferred plasmids
are pQE30, pET or pCAL or their artificial modifications (DNA
fragments obtained by treating pQE30, pET or pCAL with suitable
restriction endonucdeases) for E. coli; pYES2 or pPIC9K for yeasts;
and pBacPAK8/9 for insect cells.
[0071] Enhancer sequences and terminator sequences may be those
commonly used by those skilled in the art such as those derived
from SV40.
[0072] Conventional selectable markers can be used by standard
methods. Examples are genes resistant to antibiotics such as
tetracycline, ampicillin, kanamycin, neomycin, hygromycin or
spectinomycin.
[0073] Expression vectors can be prepared by continuously and
circularly linking at least said promoter, start codon, gene
encoding the desired Ppnk protein, stop codon and terminator region
to a suitable replicable unit. During then, a suitable DNA fragment
(such as a linker or a restriction site) can be applied by standard
methods such as digestion with a restriction endonuclease or
ligation with T4DNA ligase, if desired.
[0074] The expression vectors can be transduced into host cells by
using known techniques. For example, bacteria (such as E. coli,
Bacillus subtilis) can be transformed by the method of Cohen et al.
[Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast
method [Mol. Gen. Genet., 168, 111 (1979)] or the competent method
[J. Mol. Biol., 56, 209 (1971)]; Saccharomyces cerevisiae can be
transformed by the method of Hinnen et al. [Proc. Natl. Acad. Sci.
USA, 75, 1927 (1978)] or the lithium method [J.B. Bacteriol., 153,
163 (1983)]; plant cells can be transformed by the leaf disc method
[Science, 227, 129 (1985)] or electroporation [Nature, 319, 791
(1986)]; animal cells can be transformed by the method of Graham
[Virology, 52, 456 (1973)]; and insect cells can be transformed by
the method of Summers et al. [Mol. Cell. Biol., 3, 2156-2165
(1983)].
[0075] Purification and isolation of the Ppnk protein of the
present invention can be accomplished by appropriately combining
conventional methods for purifying and isolating proteins, such as
ammonium sulfate precipitation, ion exchange chromatography (e.g.
DEAE-Cellulofine, MonoQ, Q Sepharose).
[0076] When the NAD.sup.+ kinase protein of the present invention
accumulates in host cells, for example, the host cells are
collected by centrifugation or filtration or the like and suspended
in a suitable buffer (e.g. a buffer such as Tris buffer, phosphate
buffer, HEPES buffer or MES buffer at a concentration of about 10
mM -100 mM and desirably in a pH range of 5.0-8.0 though the pH
depends on the buffer used), then the cells are disrupted by a
method suitable for the host cells used (e.g. ultrasonication) and
centrifuged to collect the contents of the host cells. When the
NAD.sup.+ kinase protein of the present invention is secreted
outside host cells, however, the host cells and the culture medium
are separated by centrifugation or filtration or the like to give a
culture filtrate. The host cell lysate or the culture filtrate can
be used to isolate/purify the protein directly or after ammonium
sulfate precipitation and dialysis. The isolation/purification can
be performed as follows. When the protein of interest is tagged
with 6.times.histidine, GST, maltose-binding protein or the like,
conventional methods based on affinity chromatography suitable for
each tag can be used. When the protein of the present invention is
produced without using these tags, the method based on antibody
affinity chromatography can be used, for example. These methods may
be combined with ion exchange chromatography, gel filtration or
hydrophobic chromatography, isoelectric chromatography or the
like.
Examination on Reaction Conditions for Preparing NADP
[0077] Processes for preparing NADP according to the present
invention rely on a reaction using polyphosphate and NAD.sup.+ as
substrates in the presence of a polyphosphate-dependent NAD.sup.+
kinase.
[0078]
NAD.sup.++(polyP).sub.n.fwdarw.NADP.sup.++(polyP).sub.n-1
[0079] The efficiency of the above reaction is influenced by
various conditions such as temperature, pH, the type of
polyphosphate, the concentration of polyphosphate in the reaction
solution, and the level and type of metal ions. After examining
various conditions, we found that the efficiency of the reaction is
especially influenced by the concentrations of polyphosphate and
metal ions in the reaction solution.
[0080] The concentration of polyphosphoric acid or a salt thereof
in the reaction solution is 0.1-15% by weight, preferably 2-10% by
weight, more preferably 5-10% by weight, most preferably about 10%
by weight of the reaction solution. The type of polyphosphate is
not specifically limited. For example, it can be appropriately
selected from metaphosphoric acid, hexametaphosphoric acid and the
like described-above.
[0081] The above reaction catalyzed by a polyphosphate-dependent
NAD.sup.+ kinase requires the presence of a metal ion, especially a
divalent metal ion. The concentration of the divalent metal ion in
the reaction solution is 5-150 mM, preferably 50-150 mM, more
preferably 50-100 mM, most preferably 100 mM. The divalent metal
ion is not specifically limited, but preferably selected from
magnesium ion or manganese ion. Especially preferred is magnesium
ion. The metal ion is preferably contained in the reaction solution
as a chloride, sulfate or nitrate.
[0082] In order to obtain NADP.sup.+ at a high yield after the
completion of the NAD.sup.+ kinase-mediated reaction, it is
important not only to attain a high phosphate transfer efficient
from NAD.sup.+ but also to stably maintain NADP.sup.+ once
synthesized. NADP.sup.+ is known to be less stable to heat and pH
than NAD.sup.+ (e.g. see Year 2000 Catalog of Oriental Yeast, Co.,
Ltd.). Thus, conditions for the NAD.sup.+ kinase-mediated reaction
should be selected in such a manner that NADP.sup.+ has a stable
composition.
[0083] The reaction should preferably be performed under the
conditions of, but not limited to, a temperature of 20-37.degree.
C., more preferably 37.degree. C. The pH is preferably 5-8, more
preferably 6-7. Processes of the present invention use an NAD.sup.+
kinase from Mycobacteria having an optimal working pH range in
which NADP.sup.+ is more stable (in a range of pH 3-7).
Forms of NAD.sup.+ Kinases
[0084] The NAD.sup.+ kinases used in the processes of the present
invention may be in the form of a solubilized protein purified from
natural sources or recombinant host cells, or an immobilized enzyme
obtained by immobilizing the solubilized protein. Alternatively,
cells expressing an NAD.sup.+ kinase protein may be directly
immobilized by a chemical treatment such as acrylamide and used as
immobilized enzymatic cells.
[0085] Methods for purifying NAD.sup.+ kinase proteins from natural
sources or recombinant host cells were described in detail above in
the section of "Processes for preparing NAD.sup.+ kinase proteins".
The resulting purified protein can be used as a solubilized
protein. Alternatively, the purified protein can be immobilized and
used as an immobilized enzyme. Enzymes can be immobilized by known
methods. For example, a purified NAD.sup.+ kinase protein can be
immixed with an activated gel (e.g. Formyl-Cellulofine gel (from
Chisso Co.)) under non-deactivating mild conditions to immobilize
the purified NAD.sup.+ kinase protein on the insolubilized gel.
Detailed descriptions of procedures for immobilizing purified
proteins are found in e.g. catalogs of SEIKAGAKU CORPORATION Co.
for the use of Formyl-cellulofine gel. Activated gels are also
described in many other documents such as the manufacturer's manual
of Pharmacia Biotech.
[0086] Alternatively, it is more effective to immobilize microbial
cells expressing an NAD.sup.+ kinase protein or to use a cellular
subfraction to adopt a method based on a metabolic pathway present
in the microbial cells such as a glycolytic system. Typical
examples are described in Murayama A et al. Biosci. Biotech.
Biochem. 2001 65:644-650 [14] and Fujio T et al. Biosci. Biotech.
Biochem. 1997 61:956-959 [15].
[0087] Known methods for immobilizing microbial cells use
carrageenan or acrylamide gel. Methods for immobilizing microbial
cells are described in e.g. Murata K et al. Biotechnol. Bioeng.,
1979 21:887-895, supra. [4]. Various improvements were added e.g.
by disrupting cellular membranes by a treatment with an organic
solvent such as acetone to facilitate the action of intracellular
soluble enzyme after microbial cells have been immobilized.
[0088] In Example 4 below as a non-limiting example, host cells
overexpressing Ppnk protein were immobilized in a polyacrylamide
gel matrix and treated with acetone to increase the permeability of
the substrates [NAD and polyphosphate (poly(P).sub.n)] and/or
products [NAD and polyphosphate (poly(P).sub.n-1)].
[0089] Specifically, harvested host cells were first washed with
cooled saline and then immobilized by the method described in [10]
with some modifications. Host cells are first suspended in 0.75M
Tris-HCl (pH 8.8) and thoroughly mixed with an acrylamide solution,
then left in ice water at 0-4.degree. C. for 10 minutes to 1 hour.
The acrylamide solution comprises e.g. 10-50% acrylamide, 0.2-1%
N,N'-methylenebisacrylamide, 0.1-0.5%
N,N,N',N'-tetramethylethylenediamine, and 0.1-0.5% potassium
ammonium persulfate. The resulting gel is cut into cubes (e.g., 3.0
mm.times.3.00 mm.times.3.0 mm).
[0090] The above gel may be further treated with acetone.
Specifically, the cubic gel is suspended in acetone and incubated
with mild stirring at 0.degree. C. for 2 minutes to 20 minutes. The
gel is thoroughly washed with cooled 5.0 mM Tris-HCl buffer (pH
7.0) and stored in a similar buffer containing e.g. 0.01 mM to 1 mM
NAD.sup.+ and 0.01 mM to 10 mM MgCl.sub.2 at about 4.degree. C.
before use.
[0091] In Example 4, 5.0g (wet weight) of SK27 cells or SK45 cells
were immobilized in 15 ml of polyacrylamide gel, i.e. in a ratio of
about 0.33g (wet weight) of cells/ml gel.
[0092] NADP production by using purified Ppnk protein has several
disadvantages. For example, (i) lengthy and complex operations are
required for purifying Ppnk protein, (ii) Ppnk is not very stable,
and (iii) the enzyme cannot be reused unless it is insolubilized.
In contrast, immobilized cells can be conveniently used because no
purification of the enzyme is required and they can be applied to
substrate solutions having higher ion strength. The immobilized
cells have also the advantage that the enzyme can be reused because
it can be readily separated from the reaction solution by
filtration or other means after the completion of the reaction.
[0093] However, the immobilized cells have the following
disadvantages: the amount of NAD.sup.+ kinase present per unit
cells is insufficient and the enzyme tends to be partially
deactivated by chemical treatment during preparation of the
immobilized cells. This invites problems such as the low production
yield of NADP.sup.+ even after a continuous enzymatic reaction for
a long period (e.g. 2-7 days), generation of decomposition products
by the extended reaction and coloration of the mixed solution after
the completion of the reaction. These disadvantages do not occur
when purified NAD.sup.+ kinase proteins are used.
[0094] The present invention enabled NADP production on a
commercial scale by adopting appropriate reaction conditions
whichever the purified proteins, the immobilized enzymes or
immobilized cells is used.
References
[0095] 1. Beutler, H. O. and Supp, M.: Coenzymes, metabolites, and
other biochemical reagents, pp. 328-393. In Bergmeyer, H. U. (ed.),
Methods of enzymatic analysis. Vol, 1. Verlag Chemie, Weinheim
(1983).
[0096] 2. Matsushita, H., Yokoyama, S., and Obayashi, A.: NADP
production using thermostable NAD kinase of Corynebacterium
flaccumfaciens AHU-1622. Can. J. Microbiol., 32, pp. 585-590
(1986).
[0097] 3. McGuinnes, E. T. and Bulter, J. R.: NAD kinase- A review.
Int. J. Biochem., 17, pp. 1-11 (1985).
[0098] 4. Murata, K., Kato, J., and Chibata, I.: Continuous
production of NADP by immobilized Brevibacterium ammoniagenes
cells. Biotechnol. Bioeng., 21, pp. 887-895 (1979).
[0099] 5. Murata, K., Uchida, T., Tani, K., Kato, J., and Chibata,
I.: Metaphosphate: A new phosphoryl donor for NAD phosphorylation.
Agric. Biol. Chem., 44, pp. 61-68 (1980).
[0100] 6. Wood, H. G. and Clark, J. E.: Biological aspects of
inorganic polyphosphates. Ann. Rev. Biochem., 57, pp. 235-260
(1988).
[0101] 7. Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto,
W., Yamada, T., and Murata, K.: Inorganic polyphosphate/ATP-NAD
kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv.
Biochem. Biophys. Res. Commun., 276, pp. 57-63 (2000).
[0102] 8. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,
Seidman, J. G., Smith, J. A., and Struhl, K.: Current Protocols in
Molecular Biology. John Wiley & Sons, Inc., New York
(1994).
[0103] 9. Bradford, M.: A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein dye-binding. Anal. Biochem., 72, pp. 248-254
(1976).
[0104] 10. Chibata, I., Tosa, T., and Sato, T.: Immobilized
aspartase-containing microbial cells: preparation and enzymatic
properties. Appl. Microbiol., 27, pp. 878-885 (1974).
[0105] 11. Murata, K., Uchida,T., Tani,K., Kato,J.,and Chibata,I.:
Continuous production of glucose-6-phosphate by immobilized
Achromobacter butyri cells. Eur. J. Appl. Microbiol. Biotechnol.,
7, pp. 45-50 (1979).
[0106] 12. Fiske, C. H. and Subbarow, Y.: The colorimetric
determination of phosphorus. J. Biol. Chem., 66, pp. 375-400
(1925).
[0107] 13. Langer, R. S., Hamilton, B. K., Gardner, C. R., Archer,
M. C., and Colton, C. K.: Enzymatic regeneration of ATP. AIChE J.,
22, pp. 1079-1090 (1976).
[0108] 14. Maruyama, A. and Fujio, T.: ATP-production from adenine
by a self-coupling enzymatic process: high-level accumulation under
ammonium-limited conditions. Biosci. Biotech. Biochem., 65, pp.
644-650 (2001).
[0109] 15. Fujio, T. and Maruyama, A.: Enzymatic production of
pyrimidine nucleotides using Corynebacterium ammoniagenes cells and
recombinant Escherichia coli cells: Enzymatic production of
CDP-choline from orotic acid and choline chloride (Part I). Biosci.
Biotech. Biochem., 61, pp. 956-959 (1997).
[0110] 16. Kawai, S., Mori, S., Mukai, T., Hashimoto, W., and
Murata, K.: Molecular characterization of Escherichia coli NAD
kinase. Eur. J. Biochem., in press (2001).
[0111] 17. Kawai, S., Suzuki, S., Mori, S., and Murata, K.:
Molecular cloning and identification of UTR1 of a yeast
Saccharomyces cerevisiae as a gene encoding an NAD kinase. FEMS
Microbiol. Lett., in press (2001).
[0112] 18. Waehneldt, T.V. and Fox, S.: Phosphorylation of
nucleosides with polyphosphoric acid. Biochim. Biophys. Acta, 134,
pp. 9-16 (1967).
[0113] 19. S. T. Cole, et al.,: Deciphering the biology of
Mycobacterium tuberculosis from the complete genome sequence.
Nature 393, pp. 537-544 (1998).
[0114] 20. "Enzyme Handbook" 1983, Asakura Publishing, p. 339.
[0115] 21. Brigitte E.et al., Method in Enzymology 185: pp. 94-103
(1990).
[0116] 22. Suzuki Y. et al., J. Bacteriol., 169, pp. 839-843
(1987).
EXAMPLES
[0117] The following examples further illustrate the present
invention without, however, limiting the technical scope of the
invention thereto. Various changes and modifications can be added
to the invention by those skilled in the art on the basis of the
description herein, and such changes and modifications are also
included in the technical scope of the invention. Unless otherwise
indicated, the following methods were used in the examples
herein.
[0118] 1) Assay for the Polyphosphate-dependent NAD Kinase Activity
of Ppnk
[0119] The polyphosphate-dependent NAD kinase activity of Ppnk was
assayed in a reaction mixture (1.0 ml) containing 5.0 mM NAD, 5.0
mM MgCl.sub.2, 100 mM Tris-HCl (pH 7.0), 1.0 mg/ml polyphosphate
and Ppnk protein as previously described [5], [7]. The
polyphosphate used was metaphosphoric acid (Wako Pure Chemical
Industries, Osaka, Japan). One unit of Ppnk activity was defined as
the activity of producing 1.0 .mu.mol of NADP at 37.degree. C. in 1
hour. The specific activity was expressed in units/mg protein. The
protein level was determined by using the bovine serum albumin as a
standard according to the method of Bradford et al. [9].
[0120] (2) Assay for NADP-producing Activity
[0121] A reaction for producing NADP was performed in a reaction
mixture solution (4.0 ml ) at 37.degree. C. with shaking. The
reaction mixture solution consists of 50 mM NAD, 100 mM MgCl.sub.2,
100 mM Tris-HCl (pH 7.0), 50 mg/ml polyphosphate (metaphosphoric
acid), and one of purified Ppnk (14.4 units, i.e. 0.16 mg protein)
or various cell preparations [cells (0.03 g) or the immobilized
cells (0.10 g) or homogenates thereof]. After the reaction for 1
hour, 10 .mu.l of the reaction mixture was collected and
enzymatically assayed for NADP using isocitrate dehydrogenase
(Sigma-Aldrich Japan, Tokyo, Japan) [7]. The activity was expressed
in .mu.mol/g cells/hour.
[0122] (3) Preparation of Homogenates
[0123] Homogenates of intact cells and acetone-treated cells were
prepared by disrupting cells with a sonifier (Branson, Danbury,
Conn.) at 0.degree. C. for 10 min in 5.0 ml of 5.0 mM Tris-HCl (pH
7.0). Homogenates of immobilized cells and acetone-treated
immobilized cells were prepared by grinding 3.0 ml of gel with a
pestle at 0.degree. C. for 20 min in 5.0 ml of 5.0 mM Tris-HCl (pH
7.0).
Example 1
Expression of a Recombinant Ppnk Protein
[0124] 1) Construction of an Expression Vector
[0125] According to the method of Kawai et al. [7], an open reading
frame of unknown function Rv1695 was amplified by PCR from M.
tuberculosis H37Rv chromosomal DNA. Specifically, chromosomal DNA
of M. tuberculosis H37Rv was first prepared from cultured cells as
described in Suzuki Y. et al., J. Bacteriol., 169, pp. 839-843
(1987) [22]. The genomic sequence of H37Rv chromosomal DNA can be
found at http://www.sanger.ac.uk/Projects/M
tuberculosis/cosmids.stml. Then, an NdeI primer having an NdeI
restriction site and a BamHI antisense primer having a BamHI
restriction site as shown below were used to specifically amplify
Rv1695 and to permit the insertion of amplified Rv1695 into a
plasmid using NdeI/BamHI sites.
[0126] NdeI primer:
[0127] 5'-ccc ata tga ccg ctc atc gca gtg ttc tg-3'
[0128] (SEQ ID NO: 3)
[0129] BamHI Antisense Primer:
[0130] 5'-cgg atc cct act ttc cgc gcc aac cgg tc-3'
[0131] (SEQ ID NO: 4)
[0132] The PCR reaction solution had the following composition (in
100 .mu.L): 133 KOD buffer (Toyobo Co., Ltd.) containing 2.5U KOD
polymerase (Toyobo Co., Ltd.), 0.25 .mu.g M. tuberculosis H37Rv
chromosomal DNA, 40pmol NdeI primer, 40 pmol BamHI antisense
primer, 20 nmol dNTPs and 100 nmol MgCl.sub.2. The PCR reaction
consisted of 25 cycles of 98.degree. C. for 15 seconds
(denaturation), 67.degree. C. for 2 seconds (annealing) and
74.degree. C. for 30 seconds (extension) to give an intended PCR
product of 0.93 kb.
[0133] The nucleotide sequence of the PCR product was determined
and verified to be identical to Rv1965 of M. tuberculosis H37Rv
(see http://www.sanger.ac.uk/Projects/M tuberculosis/cosmids.stml)
[19]. Then, the resulting NdeI/BamHI fragment of the ppnk gene
encoding Ppnk was inserted into the E. coli expression plasmid
pET3a (Novagen) under the control of the T7 promoter using
NdeI/BamHI sites to construct an expression plasmid vector.
[0134] 2) Expression of Recombinant Proteins
[0135] The above expression vector was used to transform host
cells, thereby expressing a recombinant M. tuberculosis Ppnk
protein. The host cell strain used was E. coli BL21(DE3)pLysS
(Novagen, Darmstadt, Germany) [7]. The expression vector pET3a-NADK
containing the Ppnk gene was transformed into E. coli
BL21(DE3)pLysS competent cells (Novagen) according to a known
method to give a recombinant designated SK27. The recombinant SK27,
i.e. pET3a-NADK/BL21(DE3)pLysS was deposited on Jun. 20, 2001 with
the International Patent Organism Depositary of the National
Institute of Advanced Industrial Science and Technology (residing
at Tsukuba Central 6, 1-1-1 Higashi, Tsukuba-city,
Ibaraki-prefecture, 305-8566, Japan) under the accession number
FERM P-18383. As a control, the empty vector pET3a not containing
the Ppnk gene was transformed into BL21 in the same manner to give
a recombinant designated SK45.
[0136] Similarly, the Rv1695 gene amplified by PCR was inserted
into the plasmid pTRP-2cis according to the method described in
Japanese Patent Public Disclosure No. 103278/96 and Japanese Patent
Public Disclosure No. 191984/98 and then transformed into the E.
coli JM109 competent cells (Takara Bio Inc.) to give a recombinant
designated pTRP-2cis-NADK/JM109. The transformant was deposited on
Jun. 20, 2001 with the International Patent Organism Depositary of
the National Institute of Advanced Industrial Science and
Technology under the accession number FERM P-18384.
[0137] Cell cultures of SK27 (and SK45) were incubated with shaking
in LB liquid medium containing 100 .mu.g/mL ampicillin and 34
.mu.g/mL chloramphenicol [8] at 37.degree. C. until OD600 reached
around 0.7, at which 0.4 mM isopropyl-.beta.-D galactopyranoside
(IPTG) was added and then the cultures were cooled to 18.degree. C.
Then, incubation with shaking was continued for 3 days to induce
the expression of the recombinant Ppnk protein [7]. In the case of
pTRP-2c-Rv1695/JM109, cultures were incubated with shaking
overnight in LB liquid medium containing 100 .mu.g/mL ampicillin at
37.degree. C. to induce the expression of the recombinant Ppnk
protein.
Example 2
NAD.sup.+ Kinase Activity of Ppnk Protein
[0138] After the transformed E. coli cells of Example 1 were
disrupted, polyphosphate-dependent NAD.sup.+ kinase activity was
recovered as soluble fraction into the lysate. The activity was
tested as described in (1) Assay for the polyphosphate-dependent
NAD kinase activity of Ppnk in "Materials and Methods" to show an
activity level of about 6,000 units/L culture medium, i.e. 31
units/mg cells extracts. This is about 400 times higher than the
activity of the NAD kinase of Brevibacterium ammoniagenes (0.075
units/mg) that has been conventionally used for producing NADP from
NAD and polyphosphate (metaphosphoric acid) [4]. The NAD.sup.+
kinase activity of the recombinant Ppnk protein was assayed by the
method of Kawai S et al. supra. [7] and one unit was defined as the
activity of producing 1 .mu.mol NADP.sup.+ at 37.degree. C. in 60
minutes.
Example 3
Purification of the Recombinant Ppnk Protein
[0139] To purify the recombinant Ppnk protein, the frozen cell
cultures of Example 1 were first thawed and resuspended at 10%
(w/v) in an extraction buffer (containing 10 mM potassium phosphate
buffer (pH 7.5), 0.1 mM NAD.sup.+, 1 mM 2-mercaptoethanol and 0.5
mM EDTA). Then, the suspension was sonicated in ice water for 5
minutes. After centrifugation, the extract supernatant was
collected.
[0140] The extract was loaded onto a column which has been packed
with DEAE-Cellulofine (SEIKAGAKU CORPORATION) and has been
equilibrated with the extraction buffer, and the column was
thoroughly washed. As a result, NAD.sup.+ kinase activity could be
recovered as a single elution peak near 0.2M NaCl by gradient
elution of 0-0.5M NaCl. At this stage, the recombinant Ppnk protein
including substantially no phosphatase could be obtained. The
activity was tested as described in (1) Assay for the
polyphosphate-dependent NAD kinase activity of Ppnk in "Materials
and Methods" to show a specific activity of about 150 units/mg
protein.
[0141] Enzymatic properties of the resulting purified M.
tuberculosis Ppnk protein were evaluated and compared with the
properties of a previously known polyphosphate-dependent NAD kinase
from Micrococcus flavus. The results are shown in Table 2
below.
3TABLE 2 (Enzymatic properties of Ppnk proteins) Micrococcus flavus
M. tuberculosis H37Rv (control) (present invention) Molecular
weight 68,000 140,000 (gel filtration) Optimal temperature
55.degree. C. 50.degree. C. Optimal pH pH 7.0 pH 5.5-6.5
ATP/polyphosphate ATP (100%) ATP (100%) reaction ratio
Poly(p).sub.4(18%) Poly(p).sub.4 (121%) Polyphosphate Poly(p).sub.4
(100%) Poly(p).sub.4 (100%) specificity polyphosphate (91%)
polyphosphate (92%) metaphosphate (58%) metaphosphate (109%)
hexametaphosphate hexametaphosphate (58%) (72%) Activation by metal
MgCl.sub.2 (100%) MgCl.sub.2 (100%) ions (each 1 mM) MnCl.sub.2
(143%) MnCl.sub.2 (268%) CaCl.sub.2 (65%) CaCl.sub.2 (34%)
CoCl.sub.2 (51%) CoCl.sub.2 (55%) CuCl.sub.2 (33%) CuCl.sub.2 (8%)
ZnCl.sub.2 (30%) ZnCl.sub.2 (30%) AlCl.sub.3 (0%) AlCl.sub.3
(0%)
[0142] Comparison of enzymatic properties showed that the
recombinant Ppnk protein from M. tuberculosis H37Rv is a preferable
transferase for synthesizing NADP.sup.+ from NAD.sup.+ using
polyphosphate as a phosphate donor.
Example 4
Immobilization of Cells into a Polyacrylamide
[0143] NADP production by using purified Ppnk protein has several
disadvantages:
[0144] (i) lengthy and complex operations are required for
purifying Ppnk,
[0145] (ii) Ppnk is not very stable, and
[0146] (iii) the enzyme cannot be reused (unless it is
insolubilized).
[0147] Ppnk protein was immobilized on an ion exchanger and used
for continuous production of NADP from NAD and metaphosphoric acid.
Immobilized enzyme (Ppnk) systems are currently available on a
commercial scale. However, it is thought that immobilized cells can
be more conveniently used than the immobilized enzyme because no
purification of the enzyme is required and substrate solutions
having higher ion strength can be applied. Thus, SK27 cells
overexpressing Ppnk protein were entrapped in a polyacrylamide gel
matrix and treated with acetone to increase the permeability of the
substrates [NAD and polyphosphate (poly(P).sub.n)] and/or products
[NADP and polyphosphate (poly(P).sub.n-1)].
[0148] Specifically, SK27 or SK45 cells cultured in Example 1 were
first harvested and washed twice with 0.85% cooled saline. The
cells were immobilized according to the method described in [10]
with some modifications.
[0149] Specifically, 5.0 g (wet weight) of SK27 or SK45 cells were
suspended in 6.0 ml of 0.75M Tris-HCl (pH 8.8). The cell suspension
was thoroughly mixed with 4.5 ml of an acrylamide solution (30%
acrylamide, 0.6% N,N'-methylenebisacrylamide, 0.25%
N,N,N',N'-tetramethylethylenediam- ine, and 0.25% potassium
ammonium persulfate), and then, left at 0.degree. C. for 30
minutes. The resulting gel (15 ml) was cut into cubes (3.0
mm.times.3.00 mm.times.3.0 mm).
[0150] This was further suspended in 50 ml of acetone and incubated
at 0.degree. C. for 5 minutes with mild stirring. The gel was
washed twice with cooled 5.0 mM Tris-HCl buffer (pH 7.0) and stored
in 5.0 mM Tris-HCl buffer (pH 7.0) containing 0.10 mM NAD and 0.10
mM MgCl.sub.2 at 4.degree. C. before use.
[0151] By this method, 5.0 g (wet weight) of SK27 or SK45 cells
were immobilized in 15 ml of polyacrylamide gel, i.e. in a ratio of
about 0.33 g (wet weight) of cells/ml gel.
Example 5
NADP Production by Purified Ppnk Protein
[0152] Ppnk (150 units/mg) purified from cell extracts of SK27 [7]
was used to produce NADP. The NADP-producing activity was assayed
using 14.4 units of purified Ppnk in the presence of 50 (solid
squares), 100 (solid circles) or 150 (solid triangles) mg/ml of
metaphosphoric acid according to the method described in (2) Assay
for NADP-producing activity in "Materials and Methods". The results
showed that 30 mM (27 g/l) NADP was produced from 50 mM NAD and 100
mg/ml metaphosphoric acid (FIG. 2A). However, the transfer from NAD
to NADP remained less than 60% irrespective of the concentration of
metaphosphoric acid (FIG. 2A).
[0153] The low transfer efficiency is attributed to the fact that
the polyphosphate-dependent NAD kinase activity of Ppnk protein was
inhibited by the NADP produced. In fact, the
polyphosphate-dependent NAD kinase activity of Ppnk is
significantly inhibited by NADP but not inhibited by ADP. The
inhibition is substantially completely induced by 30 mM NADP (FIG.
2B).
Example 6
NADP-producing Activity of Various Cell Preparations
[0154] Various cell preparations were tested for the NADP-producing
activity as described in (2) Assay for NADP-producing activity in
"Materials and Methods". The results are shown in Table 3
below.
4TABLE 3 NADP-producing activity of various cell preparations
NADP-producing activity Cell preparation (.mu.mol/g cells/hour)
Intact cells 93.5 Homogenate of intact cells (p) 1530
Acetone-treated cells 642 Homogenate of acetone-treated cells 1420
Immobilized cells 146 Homogenate of immobilized cells (q) 925
Acetone-treated immobilized cells 672 Homogenate of acetone-treated
843 immobilized cells
[0155] The NADP-producing activity of the cell homogenate was 1,530
.mu.mol/g cells/hour. This is defined as "p". On the other hand,
the NADP-producing activity of the homogenate of the immobilized
cells was 925 .mu.mol/g cells/hour. This is defined as "q". This
means that about 60% [(q/p).times.100] of the NADP-producing
activity initially present in the intact cells was incorporated
into the polyacrylamide gel. It should also be noted that the
activity of the homogenate of the acetone-treated immobilized cells
(843 .mu.mol/g cells/hour) was higher than that of the
acetone-treated immobilized cells (672 .mu.mol/g cells/hour). This
suggests that the polyacrylamide gel matrix may hinder the transfer
of substrates and/or products.
[0156] The acetone-treated immobilized cells used here were those
obtained in Example 4. The immobilized cells without acetone
treatment were the cells before acetone treatment in Example 4. The
acetone-treated cells not immobilized on polyacrylamide were
obtained by washing cells before acrylamide treatment in ice-cold
5.0 mM Tris-HCl buffer (pH 7.0) and collecting them, followed by
acetone treatment as described in Example 4.
Example 7
Functions of Ppnk in the Immobilized Cells
[0157] Functions of Ppnk in the acetone-treated immobilized cells
obtained in Example 4 were evaluated and compared with those in the
non-immobilized acetone-treated cells by the method described in
(2) Assay for NADP-producing activity in "Materials and Methods"
unless otherwise indicated below.
[0158] (1) Effect of Immobilization
[0159] A heat treatment at 60.degree. C. for 10 minutes was
required to deactivate 50% of the NADP-producing activity of Ppnk
in the acetone-treated immobilized cells. While, a treatment at
50.degree. C. sufficed to deactivate 50% of the activity of Ppnk in
the non-immobilized acetone-treated cells (FIG. 3A). This shows
that the thermostability of Ppnk is enhanced by immobilization into
a polyacrylamide gel matrix. The optimal temperature for the
NADP-producing activity of Ppnk transferred from 50.degree. C. to
55.degree. C. by immobilization (FIG. 3B).
[0160] (2) Effect of pH
[0161] The optimal pH for Ppnk-mediated NADP production in the
acetone-treated immobilized cells was 7.0, which was somewhat
higher than the optimal pH 6.5 in non-immobilized acetone-treated
cells (FIG. 3C).
[0162] (3) Stability After Use
[0163] The acetone-treated immobilized cells were repeatedly used
in the NADP-producing reaction assay to compare the stability of
the NADP-producing activity of Ppnk after use with that of Ppnk in
the non-immobilized acetone-treated cells (FIG. 3D) under the same
conditions. The Ppnk activity in the non-immobilized cells was
wholly lost after 5 repeated runs. However, the activity in the
immobilized cells was unchanged from the start of the assay. The
half-life of the Ppnk activity in the acetone-treated immobilized
cells was estimated as 75 days or more.
Example 8
Production of NADP by Immobilized Cells
[0164] The acetone-treated immobilized cells obtained by treating
the immobilized cells with acetone which exerts NAD.sup.+ kinase
activity from the cells entrapped in acrylamide in Example 4 were
used to examine conditions for producing NADP by such cells (FIG.
4A, B, C, D) according to the method described in (2) Assay for
NADP-producing activity in "Materials and Methods" unless otherwise
indicated below.
[0165] (1) Concentration of Metaphosphoric Acid
[0166] The NADP-producing activity of Ppnk in the acetone-treated
immobilized cells increased with the concentration of
metaphosphoric acid at low levels, and reached a plateau at 100
mg/ml, and then gradually decreased (FIG. 4A). In the case of ATP,
a similar activity-substrate concentration relationship was
observed and the NADP-producing activity reached the maximum level
at 150 mM ATP. [ATP]:[Mg.sup.2+]=3:2 (FIG. 4B).
[0167] (2) NAD Levels
[0168] The NADP-producing activity of Ppnk in the acetone-treated
immobilized cells increased with the amount of NAD (FIG. 4C).
However, the activity was not determined at NAD levels higher than
50 mM.
[0169] (3) Metal Ion Levels
[0170] Mg.sup.2+ was the most effective among the metal ions
reported to be effective for the polyphosphate-dependent NAD kinase
activity of Ppnk (Mg.sup.2+, Mn.sup.2+, and Ca.sup.2+) [7]. The
highest activity was obtained at 100 mM Mg.sup.2+ in the presence
of 100 mg/ml metaphosphoric acid and 50 mM NAD (FIG. 4D). Mn.sup.2+
and Ca.sup.2+ form precipitates at concentrations above 5.0 mM.
Example 9
Examination of Reaction Conditions for Synthesizing NADP.sup.+
using M. Tuberculosis Recombinant Ppnk Protein
[0171] Both the M. tuberculosis recombinant Ppnk protein obtained
in Example 3 and the acetone-treated immobilized cells showing
NAD.sup.+ kinase activity from the cells entrapped in acrylamide
obtained in Example 4 were used to further examine optimization of
various reaction conditions for NADP.sup.+ synthesis. The results
show that optimal reaction conditions include the pH and
polyphosphate and metal ion levels shown in the table below.
5TABLE 4 Immobilized Purified enzyme enzyme (soluble) Working pH
range pH 6-8 pH 5.5-8 Level of metaphosphoric 2-15% (w/v) 2-15%
(w/v) acid (and metaphosphates) Metal ion level 50-150 mM
MgCl.sub.2 50-150 mM MgCl.sub.2
Example 10
NAD.sup.+ Kinase Activity Under Optimal Conditions of the Present
Invention
[0172] The acetone-treated immobilized cells (10 units) of Example
4 were incubated in an optimal reaction mixture [50 mM NAD, 100
mg/ml metaphosphoric acid (or 150 mM ATP (FIG. 5B)), 100 mM
MgCl.sub.2, and 100 mM Tris-HCl (pH 7.0)]. After completion of the
reaction, the amount of NADP.sup.+ synthesized was analyzed by an
enzymatic assay using an NADP.sup.+-specific glucose-6-phosphate
dehydrogenase (from yeast available from Oriental Yeast, Co.,
Ltd.). The results show that a maximum yield of 16 mM NADP was
produced (14 g/l) (FIG. 5A). However, the immobilized SK45 cells
produced no or very little NADP under the same conditions (FIG. 5A,
B).
[0173] The low conversion efficiency from NAD into NADP (about 30%)
may be attributed, but not limited, to the inhibitory effect of the
produced NADP (FIG. 2B) and/or the limitation of diffusion of the
product or substrate by the polyacrylamide gel matrix because the
decomposition of NAD and NADP by the acetone-treated immobilized
cells is negligible. Removal of NADP from the reaction system
results in an increase in transfer efficiency.
[0174] The amount of NADP produced by the immobilized SK27 cells
(16 mM, 14 g/l) (FIG. 5A) was about 8 times higher than the amount
of NADP obtained from the immobilized B. ammoniagenes cells (2.0
mM, 1.7 g/l) [4]. This value was approximately comparable to the
amount of NADP produced by using ATP (150 mM) (FIG. 5B) in place of
metaphosphoric acid as a substrate.
[0175] The NADP-producing activity of purified Ppnk protein was
also tested under optimal conditions to show that it was 30 mM, 26
g whether NADP or ATP was used. The amount of NADP.sup.+ produced
after completion of each recombinant NAD.sup.+ kinase reaction is
shown in the table below.
6TABLE 5 Ppnk protein ATP Metaphosphoric acid M. tuberculosis Ppnk
(purified) 30 mM 30 mM M. tuberculosis PpnK 16 mM 16 mM
(immobilized) (Control) Brevibacterium ammoniagenes 2 mM*)
(immobilized)
[0176] Table 5 above shows that M. tuberculosis Ppnk of the present
invention (as purified protein and/or in the immobilized
recombinant cells) is very efficient as compared with conventional
ATP-dependent NAD kinases [4].
Example 11
Analysis of Reaction Products
[0177] Purified Ppnk and immobilized cells were used for a reaction
of producing NADP in the presence of ATP or metaphosphoric acid to
examine changes of components before and after the reaction.
[0178] Specifically, an NADP-producing reaction was performed as
described in (2) Assay for NADP-producing activity in "Materials
and Methods" using the purified Ppnk protein and the immobilized
cells in the presence of 50 mg/ml metaphosphoric acid or 50 mM ATP.
At the end of the reaction for 24 hours, the reaction solution was
diluted 50-fold with 50 mM Tris-acetate buffer (pH 7.5) and the
dilution (30 .mu.l) was applied onto a TSK-GEL 80TS column (0.46 cm
in diameter.times.15 cm in height) (Tosoh, Tokyo, Japan). Then, the
nucleotide fraction adsorbed to the column was separated by
gradient elution of 0-10% methanol. The flow rate was adjusted to
0.7 ml/min. The extracted nucleotide fraction was determined by
measuring the absorbance at 260 nm.
[0179] The results show that the mixture after the reaction using
ATP and the purified Ppnk protein contained unreacted ATP and NAD
in addition to the reaction products NADP and ADP (FIG. 6B). When
ATP and the immobilized cells were used, the mixture after the
reaction further contained a decomposition product of ADP, AMP in
addition to NADP and ADP and unreacted ATP and NAD (FIG. 6C).
Thin-layer chromatography further showed that adenosine was formed
(data not shown). When metaphosphoric acid was used in place of
ATP, however, the mixture after the reaction contained only NADP
and unreacted NAD in either event the purified Ppnk (FIG. 6E) or
the immobilized cells (FIG. 6F) was used.
Example 12
Phosphate-Donating Substrates in Metaphosphoric Acid
[0180] Metaphosphoric acid is a mixture of cyclic and/or linear
polyphosphates having various degrees of polymerization. To
identify essential phosphate-donating substrates for the
polyphosphate-dependent NAD kinase activity of Ppnk, polyphosphates
in metaphosphoric acid were separated on an ion exchange column as
follows.
[0181] Metaphosphoric acid (10%, 60 ml, pH 7.0) was dialyzed
against 3,000 ml of water using a Seamless Cellulose Tube (cut-off:
12,000-14,000 Da) (Viskase Sales Corp, Chicago, Ill.) at 25.degree.
C. for 24 hours. The dialysate (2,900 ml) was loaded onto a Dowex
1.times.2 (Cr.sup.-, 200-400 meshes) column (3.0.times.6.0 cm)
(Muromachi Chemicals Ltd., Tokyo, Japan). Then, adsorbed
polyphosphates were eluted with a linear gradient of LiCl (600 ml,
0-1.0M, pH 2.0) to give a 6.0 ml fraction every 6 minutes. A part
of each fraction (0.10 ml) was used to assay the polyphosphate
dependent NAD kinase activity of Ppnk as described above. FIG. 7
shows elution patterns of phosphate donors in metaphosphoric acid
on Dowex 1.times.2 column. Solid circles represent
phosphate-donating activity, open circles represent acid-labile
phosphate, and the dotted line represents the concentration of
LiCl. The amount of NADP produced in one minute was defined as
phosphate-donating activity. Acid-labile phosphate in each fraction
was estimated by determining inorganic orthophosphoric acid
released from metaphosphoric acid after the eluate was boiled in 1N
HCl for 7 minutes [12].
[0182] As shown in FIG. 7, phosphate-donating activity was detected
in all the fractions eluted at concentrations higher than 0.20M
LiCl (fraction # 25-80 in FIG. 8), and about 84% of the
phosphate-donating activity was recovered from the dialysate when
metaphosphoric acid was dialyzed against water. This means that
most substrates for the enzyme mostly consist of polyphosphates
having a molecular weight less than about 12,000-14,000 Da. In FIG.
7, four peaks (fractions 32-40, fractions 44-52, fractions 56-60,
and fractions 64-72) are observed, suggesting that metaphosphoric
acid has at least four phosphate-donating substrates for the
polyphosphate-dependent NAD kinase.
EFFECTS OF THE INVENTION
[0183] Industrial applications of microbial enzymes have been so
far limited to the catalysis of decompositions and simple
transformation reactions. The enzymes have not been widely applied
to synthetic reactions demanding energy (ATP) supply on a
commercial scale. One limitation of the development of economically
feasible ATP-demanding processes is the lack of an appropriate
system for (re)generating and/or recycling ATP.
[0184] Therefore, it is essential to construct a system for
(re)generating ATP not only for economical utilization of the
enzyme but also for the economy of processes and the efficiency of
the reaction. For this purpose, various approaches for
(re)generating ATP have been proposed including chemical synthesis,
whole cell, cell organeller or sub-cellular systems, and cell-free
systems [13]. However, the technical and economical feasibility of
these approaches as ATP-regenerating systems has been unknown
except for the use of subcelluar systems (glycolytic systems)
despite the recent great development in technology [14][15].
[0185] The production system using polyphosphates as phosphate
donors provided by the present invention offers an alternative to
systems using ATP-dependent NAD.sup.+ kinases for producing useful
compounds for the following reasons.
[0186] 1. Polyphosphates used in the processes of the present
invention can be purchased at low prices. Accordingly, they can be
used in a sufficient amount as substrates for
polyphosphate-dependent NAD kinases (FIG. 7), so that the
production system of the present invention is very economical.
[0187] 2. According to the production system of the present
invention, the product (NADP) can be easily isolated because no
decomposition products of ATP (ADP, AMP) are contained after the
reaction (FIG. 6).
[0188] 3. Various enzymes using polyphosphates as energy sources
are found in microorganisms [6]. Some of them can be readily
applied to biosynthetic systems for producing useful biochemical
compounds (e.g. producing glucose-6-phosphate from glucose and
metaphosphate on an immobilized Achromobacter butyri cell column
[11]). Therefore, the NADP.sup.+ production reaction of the present
invention can be further combined with e.g. a glucose-6-phosphate
production reaction and NADP.sup.+-dependent glucose-6-phosphate
dehydrogenase (e.g. from yeast available from Oriental Yeast, Co.,
Ltd.) to readily synthesize NADPH from glucose, polyphosphate and
NAD.sup.+ at low cost as shown by the formulae below. 2 ( polyP ) n
+ NAD + ( polyP ) n - 1 + NADP + ( polyP ) n - 1 + glucose ( polyP
) n - 2 + glucose - 6 - phosphate glucose - 6 - phosphate + NADP +
6 - phosphogluconolactone + NADPH ( polyP ) n + NAD + + glucose (
polyP ) n - 2 + 6 - phosphogluconolactone + NADPH
[0189] 4. Genetic engineering and protein engineering techniques
well known to those skilled in the art can be used to convert known
ATP-dependent NAD.sup.+ kinases into polyphosphate-dependent
kinases. Recent findings show that the ATP-dependent NAD kinase in
E. coli also has polyphosphate-dependent NAD kinase activity but at
a very low level. The nucleotide sequences of the ATP-dependent NAD
kinases from E. coli and the yeast Saccharomyces cerevisiae [17]
are similar to the sequence of the polyphosphate/ATP-dependent NAD
kinase from M. tuberculosis H37Rv of the present invention (SEQ ID
NO: 1).
[0190] Considering that biochemical energy carriers are derived
from polyphosphates [18], the similarity of the nucleotide
sequences suggests that ATP-dependent NAD kinases were evolved from
polyphosphate-dependent NAD kinases probably by accumulation of
point mutations. In fact, NAD kinases using polyphosphates in place
of ATP as substrates are being successfully prepared by random
mutation. Various ATP-dependent NAD kinases from Mycobacteria or
other genera can be converted into polyphosphate-dependent NAD
kinases by mutation and applied to the present invention.
Sequence CWU 1
1
4 1 307 PRT Mycobacteria tuberculosis H37Rv 1 Met Thr Ala His Arg
Ser Val Leu Leu Val Val His Thr Gly Arg 1 5 10 15 Asp Glu Ala Thr
Glu Thr Ala Arg Arg Val Glu Lys Val Leu Gly 20 25 30 Asp Asn Lys
Ile Ala Leu Arg Val Leu Ser Ala Glu Ala Val Asp 35 40 45 Arg Gly
Ser Leu His Leu Ala Pro Asp Asp Met Arg Ala Met Gly 50 55 60 Val
Glu Ile Glu Val Val Asp Ala Asp Gln His Ala Ala Asp Gly 65 70 75
Cys Glu Leu Val Leu Val Leu Gly Gly Asp Gly Thr Phe Leu Arg 80 85
90 Ala Ala Glu Leu Ala Arg Asn Ala Ser Ile Pro Val Leu Gly Val 95
100 105 Asn Leu Gly Arg Ile Gly Phe Leu Ala Glu Ala Glu Ala Glu Ala
110 115 120 Ile Asp Ala Val Leu Glu His Val Val Ala Gln Asp Tyr Arg
Val 125 130 135 Glu Asp Arg Leu Thr Leu Asp Val Val Val Arg Gln Gly
Gly Arg 140 145 150 Ile Val Asn Arg Gly Trp Ala Leu Asn Glu Val Ser
Leu Glu Lys 155 160 165 Gly Pro Arg Leu Gly Val Leu Gly Val Val Val
Glu Ile Asp Gly 170 175 180 Arg Pro Val Ser Ala Phe Gly Cys Asp Gly
Val Leu Val Ser Thr 185 190 195 Pro Thr Gly Ser Thr Ala Tyr Ala Phe
Ser Ala Gly Gly Pro Val 200 205 210 Leu Trp Pro Asp Leu Glu Ala Ile
Leu Val Val Pro Asn Asn Ala 215 220 225 His Ala Leu Phe Gly Arg Pro
Met Val Thr Ser Pro Glu Ala Thr 230 235 240 Ile Ala Ile Glu Ile Glu
Ala Asp Gly His Asp Ala Leu Val Phe 245 250 255 Cys Asp Gly Arg Arg
Glu Met Leu Ile Pro Ala Gly Ser Arg Leu 260 265 270 Glu Val Thr Arg
Cys Val Thr Ser Val Lys Trp Ala Arg Leu Asp 275 280 285 Ser Ala Pro
Phe Thr Asp Arg Leu Val Arg Lys Phe Arg Leu Pro 290 295 300 Val Thr
Gly Trp Arg Gly Lys 305 307 2 921 DNA Mycobacteria tuberculosis
H37Rv 2 atgaccgctc atcgcagtgt tctgctggtc gtccacaccg ggcgcgacga
agccaccgag 60 accgcacggc gcgtagaaaa agtattgggc gacaataaaa
ttgcgcttcg cgtgctctcg 120 gccgaagcag tcgaccgagg gtcgttgcat
ctggctcccg acgacatgcg ggccatgggc 180 gtcgagatcg aggtggttga
cgcggaccag cacgcagccg acggctgcga actggtgctg 240 gttttgggcg
gcgatggcac ctttttgcgg gcagccgagc tggcccgcaa cgccagcatt 300
ccggtgttgg gcgtcaatct gggccgcatc ggctttttgg ccgaggccga ggcggaggca
360 atcgacgcgg tgctcgagca tgttgtcgca caggattacc gggtggaaga
ccgcttgact 420 ctggatgtcg tggtgcgcca gggcgggcgc atcgtcaacc
ggggttgggc gctcaacgaa 480 gtcagtctgg aaaagggccc gaggctcggc
gtgcttgggg tggtcgtgga aattgacggt 540 cggccggtgt cggcgtttgg
ctgcgacggg gtgttggtgt ccacgccgac cggatcaacc 600 gcctatgcat
tctcggcggg aggcccggtg ctgtggcccg acctcgaagc gatcctggtg 660
gtccccaaca acgctcacgc gctgtttggc cggccgatgg tcaccagccc cgaagccacc
720 atcgccatcg aaatagaggc cgacgggcat gacgccttgg tgttctgcga
cggtcgccgc 780 gaaatgctga taccggccgg cagcagactc gaggtcaccc
gctgtgtcac gtccgtcaaa 840 tgggcacggc tggacagtgc gccattcacc
gaccggctgg tgcgcaagtt ccggttgccg 900 gtgaccggtt ggcgcggaaa g 921 3
29 DNA Artificial Sequence Description of Artificial Sequence
primer for PCR amplification 3 cccatatgac cgctcatcgc agtgttctg 29 4
29 DNA Artificial Sequence Description of Artificial Sequence
primer for PCR amplification 4 cggatcccta ctttccgcgc caaccggtc
29
* * * * *
References